Platelet activation and aggregation underlies the basic “acute event” in arterial thrombosis, including strokes, peripheral artery disease and coronary artery disease (heart attacks). In the field of molecular biology, the P2Y12 protein is found on the surface of blood platelet cells, and is an important regulator in blood clotting (Dorsam and Kunapuli, “Central role of the P2Y12 receptor in platelet activation,” J. Clin. Invest. 113 (3): 340-5 (2004)), which can lead to arterial thrombosis. P2Y12 belongs to a group of G protein-coupled purinergic receptors (Murugappa and Kunapuli, “The role of ADP receptors in platelet function,” Front. Biosci. 11: 1977-86 (2006)) and is a chemoreceptor for adenosine diphosphate (ADP) (Hollopeter et al., “Identification of the platelet ADP receptor targeted by antithrombotic drugs,” Nature 409 (6817): 202-7 (2001)), (Nicholas, “Identification of the P2Y(12) receptor: a novel member of the P2Y family of receptors activated by extracellular nucleotides”. Mol. Pharmacol. 60 (3): 416-20 (2001)). The P2Y family has several receptor subtypes with different pharmacological selectivity, which overlaps in some cases, for various adenosine and uridine nucleotides. This receptor is involved in platelet aggregation, and is a potential target for the treatment of thromboembolisms and other clotting disorders.
Adenosine-5′-diphosphate (ADP) plays a key role in platelet function, because, although ADP itself is a weak platelet agonist, when secreted from the platelet dense granules where it is stored, it amplifies the platelet responses induced by other platelet agonists. The transduction of the ADP signal involves both a transient rise in free cytoplasmic calcium mediated by the Gq-coupled P2Y1 receptor, and inhibition of adenylyl cyclase mediated by the Gi-coupled P2Y12 receptor. Concomitant activation of both the Gq and Gi pathways by ADP is necessary to elicit normal ADP-induced platelet aggregation. Activation of the Gq pathway through P2Y1 leads to platelet shape change and rapidly reversible aggregation, whereas the activation of the G1 pathway through P2Y12 elicits a slow progressive and sustained platelet aggregation not preceded by shape change. In addition to its role in ADP-induced platelet aggregation, P2Y12 mediates the potentiation of platelet secretion induced by strong agonists and the stabilization of thrombin-induced platelet aggregates. P2Y12 has a more selective tissue distribution than P2Y1, making it an attractive molecular target for therapeutic intervention.
Current drug therapy involves using irreversible P2Y12 antagonists to bind to the surface P2Y12 receptors, so that the platelets to not bind to P2Y12 agonists such as adenosine diphosphate (ADP). Platelets not bound to one of these antagonists, whether reversibly or irreversibly, will bind to ADP.
The drug clopidogrel (Plavix®) is a P2Y12 antagonist that binds to the P2Y12 receptor on the platelet surface, and is marketed as an anti-thrombotic agent. When the active part of the drug is bound to P2Y12, the usual P2Y12 agonist ADP cannot bind. When ADP is blocked from its P2Y12 binding site, platelet activation is inhibited. In responding patients, this drug is life-saving. Unfortunately, Plavix® is ineffective in about 30% of the population. The major cause of resistance is failure to activate the drug in the liver. Patients who have CYT2C19, and possibly other cytochrome alleles, do not activate Plavix. In addition, there is polymorphism in P2Y12, found on the surface of platelets in some patients, which may also cause resistance. For this reason, some patients who have the ability to produce the active metabolite are still unable to benefit from Plavix®, due to an abnormality in their platelets.
Further, there is a delay to the anti-platelet aggregating effects, due to the fact that Plavix®) must be metabolized to form the active agent. A maximum plateau of inhibition of ADP-induced platelet aggregation is observed 4-5 days after daily oral administration of 75 mg Plavix® (or 500 mg ticlopidine). However, the delayed onset of action of Plavix® can be reduced to about two to five hours with a loading dose of 300-600 mg.
Prasugrel® (Eli Lilly) is a relatively new entrant to this market. Next generation P2Y12 receptor antagonists include ticagrelor and elinogrel. Ticlopidine and clopidogrel are structurally related compounds, belonging to the thienopyridine family of ADP receptor antagonists. They are pro-drugs that are inactive in vitro, and need to be metabolized in vivo by the hepatic cytochrome P-450 1A enzymatic pathway to active metabolites, which have very short half-lives. The active metabolites irreversibly and
specifically inhibit the function of the platelet P2Y12 receptor, reproducing the platelet function abnormalities that are observed in patients who are congenitally deficient in P2Y12 and in P2Y12 knock-out mice.
There is a substantial inter-individual variability in platelet inhibition by ticlopidine and clopidogrel, mostly due to the inter-individual differences to the extent of metabolism of the pro-drug to the active metabolite. Certain individuals taking clopridogrel can have insufficient inhibition of platelet function, with a concomitant higher incidence of vascular events, though some patients can achieve a beneficial effect by increasing the dose of clopidogrel. That said, those patients who take higher doses are at risk for severe toxic effects, such as bone marrow aplasia and microangiopathic thrombocytopenia, which are thought to be dose-dependent. These toxic side effects also occur, though less frequently, with ticlopidine. Because of these limitations, there has been significant research to develop new P2Y12 antagonists. Prasugrel (2-acetoxy-5-[alpha-cyclopropylcarbonyl-2-fluorobenzyl]-4,5,6,7-tetrahydrothieno[3,2-c]pyridine), a relatively new thienopyridine compound, has a much faster onset of action than clopidogrel. Prasugrel is structurally similar to other thienopyridines. The active metabolite of Prasugrel (R-138727), a sulfhydryl compound, binds covalently and irreversibly to the platelet P2Y12 receptor via a disulfide bond. As with clopridogrel, the irreversible binding of the active metabolite permanently blocks ADP-mediated P2Y12 signaling, and inhibits both glycoprotein IIb/IIIa receptor activation and platelet aggregation.
In a cross-over study, a 60 mg loading dose of Prasugrel provided rapid and highgrade, irreversible inhibition of ADP-induced platelet aggregation even in those subjects who responded poorly to a standard loading dose of Clopidogrel. The higher potency of Prasugrel compared with Clopidogrel probably reflects more efficient conversion of the pro-drug to the active metabolite. Prasugrel (marketed by Eli Lilly in the U.S. as Effient®) has proven safe and effective, but is currently only approved for use in angioplasty patients, and is associated with an increased risk of fatal bleeding. Accordingly, patients who can benefit from Plavix® may still wish to take Plavix®, even though there is another P2Y12 antagonist on the market.
Because these agents irreversibly inhibit P2Y12 function, the inhibitory effect of thienopyridines on circulating platelets lasts for approximately 10 days (the lifespan of a circulating platelet). While this is an advantage for patients, it can represent a problem for patients who need to undergo coronary bypass surgery, because treatment with clopidogrel within 4-5 days of the procedure is associated with increased blood loss, reoperation for bleeding, increased transfusion requirements, and prolonged intensive care unit and hospital length of stay. For this reason, there has been significant research to identify anti-thrombotic agents that reversibly inhibit P2Y12 function.
In some clinical situations, inhibition of platelet aggregation by fast-acting and reversible antagonists with a short half-life might be preferable to irreversible inhibitors. Cangrelor is a selective and reversible direct inhibitor of P2Y12. In a study that directly compared the effects of clopidogrel and cangrelor administration in patients with ischaemic heart disease, cangrelor infusion at 2 and 4 μg/mL/min resulted in near complete inhibition of platelet aggregation measured at 4 min after the addition of 10 μM ADP, whereas 4 to 7 days clopidogrel treatment resulted in only approximately 60% inhibition. The short half-life of the molecule (2.6 min) results in a rapid reversal of its platelet inhibitory effect. Addition of cangrelor in vitro to blood from the clopidogrel treated patients resulted in near complete inhibition of P2Y12-dependent platelet function. It must be noted, however, that cangrelor can only be given intravenuously, which limits its use in the clinical practice, and it did not show sufficient benefit to patients in a Phase III clinical trial to warrant FDA approval.
Brilinta (Ticagrelor, marketed by Astra Zeneca) is an orally administrable, reversible P2Y12 antagonist. Brilinta belongs to the same family as cangrelor of stable ATP analogues with high affinity for P2Y12. Brilinta is currently approved for sale in Europe, though at the time of this filing was not approved for sale in the United States.
Clopidogrel was issued a black box warning from the FDA on Mar. 12, 2010, as the estimated 2-14% of the US population that have low levels of the CYP2C19 liver enzyme needed to activate clopidogrel may not get the full effect. As metabolism of Prasugrel has not been shown to be effected by the same CYP450 mutations, it remains a potentially viable agent for those who cannot benefit from Clopidogrel due to the presence of the CYP450 mutations. However, while the hypothesis that Prasugrel will work better in patients who cannot metabolize clopidogrel is appealing, it has not been verified in prospective clinical trials.
Now that alternatives to Plavix® have been approved, and generic clopidogrel bisulfate (i.e., generic equivalents of Plavix®) will be available in the near future, patients will be faced with a difficult choice—take generic, relatively low cost clopidogrel bisulfate with the concomitant risk that they will not benefit from such therapy, or take non-generic next-generation anti-thrombotic agents, and pay the higher price for the non-generic therapy. Further, if a patient takes a drug that provides little or no benefit, the patient is at an elevated risk of a severe cardiovascular event, such as a myocardial infarction.
Thus, while physicians have more than one P2Y12 inhibitor (antagonist) to choose from, it would be useful for them to have the ability to tailor the most appropriate anti-thrombotic therapy to the individual patient and risk situation. Because not every patient can benefit from every P2Y12 inhibitor, it would be useful to have a rapid and inexpensive assay to determine whether or not a patient is able to respond to Plavix® other anti-thrombotic agents.
In terms of developing an appropriate assay, there is a correlation between patients who have mutations in the Cytochrome P-450 gene, specifically, in CYT2C19, and possibly other cytochrome alleles, do not activate Plavix®, and may not benefit from clopidogrel. However, even if the assay identifies a patient as one who can metabolize Plavix®, there is polymorphism in P2Y12 that may also cause resistance, and patients who have this mutation would not be identified unless the pharmacogenomic screening also looked for these mutations.
In any event, pharmacogenomic screening assays are available to predict whether or not a patient is susceptible to this problem. However, pharmacogenomic screening is relatively expensive, and it takes a significant amount of time to obtain the results. Because the use of pharmacogenomic assays is not widespread, patients have been prescribed Plavix® who may derive no benefit from it. As a result, patients have been faced with relatively high costs, and potentially relatively little or no efficacy. From an economic perspective, roughly 30% of patients are deriving little or no benefit form Plavix®, so in the US approximately $1.6 billion/year is spent on a drug that is not appropriate for the patients. This waste could be avoided by identifying those patients who are not expected to respond favorably to this agent, or to other anti-thrombotic agents. However, as it has been estimated that the cost of a 2C19 screen is around $500, and around 100 million patients have been prescribed Plavix, the cost of screening all of these patients would be around $50 billion. Further, unless one can identify patients with a polymorphism in P2Y12 that also renders platelets non-responsive to Prasugrel, Clopidogrel, or other P2Y12 antagonists, patients might also be administered these agents, and not benefit from them.
Accordingly, a less expensive assay is needed, as is an assay that will identify patients who are non-responsive because of mutations in their CYP450 genes, or mutations in their P2Y12 receptor. Genetic screening to identify patients with the CYP 2C19*2 and *3 alleles will identify most, but not all of the patients who cannot activate the prodrug. Screening of platelets from patients taking the drug to show whether their platelets activate after exposure to the appropriate agonist is the only way to identify for certain all resistant patients.
Currently there is no effective assay to screen patients to determine with certainty whether their platelets will bind to P2Y12 antagonists, that is, whether the patient can actually metabolize the drug, and whether the active metabolite is capable of binding to the patient's platelets. If resistant patients (whether resistance is due to genetic variations in pro-drug metabolism or in the shape of the platelet P2Y12 receptor shape) could be effectively identified, it may be possible to increase the dose of Plavix in these patients and thus salvage them with a higher dose that could prove effective therapy. In addition to P2Y12, there are other receptors involved in thrombosis and platelet aggregation. These include Protease-Activated Receptor 1 (PAR1), Protease-Activated Receptor 4 (PAR4), GPIV, Thromboxane receptor (TP receptor, including TP-alpha and TP-beta), vWF antagonists, and Glycoprotein Ib (platelet), alpha polypeptide (GP1BA) also known as CD42b (Cluster of Differentiation 42b), GPIb, antagonists, and Glycoprotein (GPIIb/IIIa) antagonists.
As there would be clear benefits associated with being able to determine whether or not a patient would benefit from taking one of these anti-thrombotic agents, it would be useful to have a rapid and inexpensive assay to determine whether or not a patient will respond to a particular anti-platelet aggregation therapy. The present invention provides such an assay.